We intend to probe enzymatic reaction mechanisms from three separate but complementary routes. First we shall probe enzymatic catalyses with suicide substrates which are chemically unreactive in solution but become activated only at the active site of specific target enzymes which,recognizing them as substrates, transform them to intermediates where latent reactive functionalities can become unmasked and lead to covalent active site blocking. In addition to providing information on catalytic pathways and the identification of functional active site components, these analogs offer sufficient selectivity to offer eventual therapeutic value in vivo. By using acetylenic substrates and oxidizable alkenoic substrates we will examine flavin-dependent hydroxy acid oxidases and acyl CoA dehydrogenases, pyridoxal and coenzyme B12 dependent lysine metabolizing enzymes, transaminases, and various racemases and isomerases. We intend to examine through sterochemical and other mechanistic analysis two unusual reactions carried out by the flavoenzyme D-amino acid oxidase and the PLP-folate dependent serinehydroxymethylase. In the first instance we may gain insight into how flavoenzymes modulate reactivity of reduced coenzyme with molecular oxygen by study of an alpha beta-elimination of beta-halosubstrate. In the second case, experiments may indicate an unusual lack of sterochemical control by the serine enzyme and offer insight into the role of tetrahydofolate as aldehyde carrier. Additionally we wish to characterize the phospho-enzyme of PEP synthase and of the bacterial membrane Enzyme I of the Phosphotransferase System. The first bears on the question of pyrophosporyl-enzyme derivatives, the second on membrane transprot mechanisms. We propose the purification of the unstable enzyme I as a specifically inactive, radioactive derivative and its controlled reactivation when homogeneous.